This project is studying the acetylcholine transporter of synaptic vesicles. The transporter exchanges protons pumped into the vesicles with cytoplasmic acetylcholine (ACh) in order to store ACh for evoked release from cholinergic nerve terminals. Biochemical and molecular biological techniques will be used to probe the structure- function relationship of the vesicular ACh transporter (VAChT). The long range aim is to understand how the activity of the VAChT is regulated in the hoe that a specific pharmacology can be developed which will allow beneficial intervention in diseases of cholinergic insufficiency. The VAChT from a number of species recently was cloned. The availability of these clones will be exploited to investigate the relationship of structure to function in this transporter. The applicants wish to descover the residues that bind protons on the inside of the vesicles and move them through the membrane, the residues that bind ACh outside of the vesicles and move it through the membrane and the residues that bind vesamicol. The first aim is to raise antisera against several synthetic polypeptides specified by the cDNA sequence of the cloned rat VAChT. They will characterize the specificity of the antibodies and the side of the vesicular membrane that they bind to. The second aim is to complete sequencing of cDNA specifying the Torpedo californica VAChT so that we will know the full amino acid sequence of the VAChT, on which most biochemical work to date has been done. The third aim is to purify and sequence photo-affinity labeled peptides of the Torpedo californica VAChT that form part of the ACh and vesamicol binding sites. The fourth aim is to develop a system expressing rat VAChT under circumstances that allow quantitation of kinetic parameters and ligand-binding properties. Expression in both a mammalian cell line and in secretory pathway mutants of yeast will be tested. Expressed VAChT will be quantitatively characterized as to subcellular targeting, level of expression, ACh active transport and ligand binding. The fifth aim is to identify important residues in VAChT. Classical protein chemistry will be applied to expressed VAChT from different species and rat VAChT will be mutagenized at selected sites. The properties of the mutants will be quantitated using the expression system developed in aim 4. The resulting data will allow substantial specification of amino acids critical to specific functions in the VAChT.